I used Trizol method to extract cellular RNA, and A260/A280 was always around 2.1. The RT reagent of TAKARA and the qPCR reagent of TAKARA were used. The experimental steps were all in accordance with the reagent instructions, and the Ct value of the internal reference gene (GAPDH) of the qPCR was always around 30.
Later, it was suspected that RT was the problem, but the CT value of three batches of RT reagent was still around 30. I also verified that my qPCR reagent and primer were no problem with the cDNA obtained after reverse transcription experiment by others, and the CT value was 15.
So it should be my RT and the previous extraction operation may have problems.
So I changed a very clean experimental environment to extract RNA, and the chloroform，isopropyl alcohol and ethanol used in the extraction process of Trizol were also changed into brand new ones. As a result, the Ct value of the internal reference was changed to about 27, which was still too large.
Could you give me some advice? What else could go wrong, so that the Ct value is more than 10 cycles larger than the normal value? I can be sure that I did not add the wrong reagent in each step, because I have repeatedly checked it.
Please please please help me! ! ! I've been stuck in this RT-qPCR experiment for nearly two months.
To better understand: you cultivated some cells, then extracted the RNA by trizol, then run the RT-qPCR, right?
Has the change between 30 and 15 Cq happened after you prepared another batch of cells?
If yes, did you count the cells prior to extraction? Have you quantified the RNA prior to RT-qPCR? How many ng/ul you had of total RNA on the 30 Cq and 15 Cq experiments?
Dear Luigi Marongiu ，Thank you a lot for your attention！
15 Cq is occured when I experiment with the another positive cDNA (from another laboratoy-same trizol extraction method but different RT reagent) , and this can exclude the problems of my qPCR reagent and primer. In other words, my qPCR reagent and primer have no problems.
30 Cq is produced from the RNA I extracted, then the cDNA I got with my RT reagent .Three batches of RT reagent was tested , but the Cq was always around 30.
5 ul, 100 ng/ul total RNA was used in both 30 Cq and 15 Cq experiments.
Perhaps I have some problems in experimental steps (extraction or RT) ？ But what could have gone wrong that would have skewed the results so much？
I would be very grateful if you could give me a few pointers！
Perhaps the cells you have extracted produce less GAPDH than those from the other batch, so even if the total RNA is the same, there might be differences in the final count of GAPDH. The alternative, which is more likely, is that the RT step made the difference. The RT produces random amounts of DNA, so here you got a step that introduces a large variability.
I suggest preparing large amounts of your GAPDH standard (even by mixing different batches) and aliquot it for later use. In that way, you should always have the same Cq. But consider that DNA degrades even when frozen, so try and store DNA at high concentration, perhaps with TE to reduce degradation and improve shelf life.
Also, the Cq as such is not very indicative. It is true that the Cq of the same target should not variate more than a couple of cycles, but results in absolute or relative terms. The latter is better because takes into account the variability of the experimental setup. Record the values of the standard at each run: they should be between 2 or 3 standard deviations from the mean. Repeat runs with standard as outliers.
Anyway, if I understood correctly, the shift between 30 and 15 cq was due to the use of two different batches of GAPDH, which is expected, in part because the cells were differently collected, in part because the RT was different. The main thing is that PCR works.
Hey Wenyan Lv it would be of great help if you could share whether your problem has been resolved,if yes how it was solved as i am facing the same problem since few months.
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